WO2017200826A1

WO2017200826A1 - Assays and compounds for treatment of cancer

Info

Publication number: WO2017200826A1
Application number: PCT/US2017/032062
Authority: WO
Inventors: Evripidis Gavathiotis; Xiomaris M. COTTO-ROIS
Original assignee: Albert Einstein College Of Medicine, Inc.
Priority date: 2016-05-16
Filing date: 2017-05-11
Publication date: 2017-11-23

Abstract

Assays and compounds are disclosed for treatment of cancers.

Description

ASSAYS AND COMPOUNDS FOR TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/336,812, filed on May 16, 2016, the content of which is herein incorporated by reference into the subject application.

BACKGROUND OF THE INVENTION

[0002] Throughout this application various publications are referred to in superscripts. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.

[0003] There is a clinical need for new therapies for melanoma which is among the few cancers with a rising incidence.²⁵ Malignant melanoma affects -40,000 new patients each year in the United States and an estimated 100,000 world-wide.²⁶' ²⁷ Melanoma is an important cause of cancer among young patients (30-50 years) which increases the economic importance of the disease. The median survival time of patients with metastatic melanoma is 8.5 months, with an estimated 5-year survival of 6%.²⁸ There has been little change in these results over the past 25 years.

[0004] The mitogen-activated protein kinase (MAPK) pathway is an important driver in melanoma. MAPK is made up of several potential targets (RAS, RAF, MEK and ERK). Several inhibitors that target components and block this pathway have been developed. Some are being used while others are being evaluated in clinical trials. Unfortunately, resistance to these agents is developed and patients relapse within 6-12 months. Hence, there is a need to identify and develop new drugs for the treatment of melanoma that can block this pathway and overcome resistance to current FDA-approved treatments.

[0005] The RAS/RAF/MEK/ERK pathway is an evolutionary conserved signaling cascade, activated by growth factors, hormones and chemokines at the cell surface.¹' ² Growth factors bind to the cell surface of receptor tyrosine kinases or hormones bind to G- protein couple receptors and signal through adaptors protein such as GRB2 and exchange factors such as SOS to activate RAS.² RAS activation leads to membrane recruitment and activation of RAF proteins. Activation of RAF proteins leads to activation of MEK that subsequently activates ERK proteins. ERK proteins have many cytosolic and nuclear substrates, including transcription factors, which depending on the cellular context will mediate diverse biological functions such as cell growth, survival and differentiation.² Activating mutations of signaling components upstream or within key components of the cascade can lead to de-regulation of the pathway abnormal cell growth and tumorigenesis.³ Mutations in the RAS family and RAF family are very frequent in cancer.

[0006] The RAF family of kinase consists of three members: ARAF, BRAF, and CRAF, which are protein-serine/ threonine kinases that are related to retroviral oncogenes discovered in 1983.⁴' ⁵ Most RAF kinase protein occurs in the cytosol where the enzymes are in their dormant state.⁵ RAF regulation is highly complex and it involves several activation events such as: protein-protein interaction, RAS binding directly to the N- terminus and a number of activating phosphorylation events in the C-terminus.²' ⁵' ⁶ One key step in the activation of RAF consists in the relief of the inhibition imposed on the RAF catalytic domain by an N-terminal regulatory domain, featuring both a RAS binding domain and a cysteine-rich domain responsible for interaction with the kinase domain and for RAF auto-inhibition.⁷ Another key step in the activation of RAF proteins is the formation of homo and heterodimers within the RAF members.⁸

[0007] Among all the RAF proteins, BRAF is the family member most easily activated by RAS.⁷ BRAF exhibits higher basal kinase activity than ARAF and CRAF, which require a greater number of phosphorylation within the N-terminal region of the kinase domain to achieve full activation.⁷' ⁹' ¹⁰ Hence, potentially explaining the frequent mutational activation of BRAF in a variety of cancers, while CRAF or ARAF mutations are very rare.⁵' ⁷ The majority of BRAF mutations occur in the activation segment or in the gly cine-rich loop.¹¹ The most common substitutions are gain-of-function mutations that leads to constitutive activation of the kinase.¹¹ Over the 45 mutations that have been described for BRAF, the most frequent mutation is the substitution of valine at position 600 for glutamic acid, known as BRAF^V600E.² BRAF^V600E causes constitutive activation of the kinase as well as insensitivity to negative feedback mechanisms.¹¹' ¹²

[0008] Oncogenic BRAF^V600E accounts for almost 50% of melanoma and 0-20% of all human cancers and has been a validated therapeutic target.^{11 13} Several RAF inhibitors have been developed and some are being used while others are being evaluated in clinical trials.¹⁴ RAF kinase inhibitors effectively block MEK and ERK phosphorylation and activation in cell lines and xenografts that harbor activated mutant BRAF . ' ^" Although potent RAF kinase inhibitors have been developed and showed strong clinical efficacy, unfortunately resistance to these agents is developed and patients relapse within 6-12 months.¹⁸' ¹⁹ Several recent studies highlight RAF dimerization as the major mechanism that mediates resistance to RAF inhibitors.¹⁶'¹⁵ Three main processes that result in RAF dimerization have been demonstrated: 1) paradoxical RAF inhibitor-mediated activation of RAF signaling through RAF dimerization, 2) feedback re-activation of receptor tyrosine kinase and RAS leading to enhanced RAF dimerization and signaling, and 3) expression of a splice variant p61BRAF^V600E that lacks the region that encompasses the RAS-binding domain shows enhanced dimerization in cells with low levels of RAS activation.¹⁵'²⁰'²¹ This has led to attribute RAF dimerization as a regulatory mechanism that mediates RAF- inhibitor resistance and a major roadblock for effective disease treatment.

[0009] The present invention provides new strategies and compounds for treatment of melanoma.

SUMMARY OF THE INVENTION

[0010] This invention provides methods of treating a cancer such as a melanoma or a tumor in a subject in need thereof comprising administering to the subject ponatinib and/or hesperadin in an amount effective to treat a cancer such as a melanoma or a tumor in a subject.

[0011] The invention further provides a composition comprising ponatinib and/or hesperadin in an amount effective to treat a cancer such as a melanoma or a tumor in a subject and a pharmaceutically acceptable carrier.

[0012] The invention also provides an in-cell-western based screening assay for identifying candidate compounds for treatment of a melanoma or a tumor comprising measuring fluorescence levels of phosphorylated ERK (P-ERK) in melanoma or tumor cells harboring a constitutively active splice variant p61-BRAF^V600E dimer that is resistant to the BRAF inhibitor vemurafenib; contacting the cells with the compound in the absence of vemurafenib; and identifying the compound as a candidate compound for treatment of a melanoma or a tumor if the compound reduces P-ERK fluorescence levels to 50% or less in the absence of vemurafenib.

BRIEF DESCRIPTION OF THE FIGURES

[0013] Fig. 1A-1E. In-cell-based screening identifies Ponatinib and Hesperadin as inhibitors of the ERK signaling pathway. A-B) SKMEL239 C4 resistant melanoma cells left untreated or treated with increasing concentration of the BRAF inhibitor Vemurafenib (PLX4032) for 3hrs then assay for in-cell-western. A) Percent of phospho-ERKl fluorescence levels in cells, calculated by taking the total fluorescence levels of phosphorylated-ERKl (P-ERK1) antibody staining divided by the total fluorescence levels of ERK1 antibody staining. B) Percent of P-ERK1 fluorescence levels in cells normalized to untreated cells. C) SKMEL239 C4 resistant melanoma cells left untreated or treated with increasing concentration of Trametinib (GSK1 120212) for 3hrs, assay for in-cell-western and graphed as in A-B. D) Screening of 200 Selleck Chemical kinase inhibitors. SKMEL239 C4 melanoma resistant cells left untreated (regular media), treated with 0.5μΜ Vemurafenib, 0.1 μΜ Trametinib or with 5μΜ of each kinase inhibitors in the presence of 0.5μΜ Vemurafenib (PLX4032) for 3hrs, assay for in-cell-western and graph as in A-B. E) SKMEL239 C4 melanoma cells left untreated (regular media), treated with 0.5μΜ Vemurafenib, 0.1 μΜ Trametinib or with 5μΜ Ponatinib or 5μΜ Hesperadin in the presence or absence of 0.5μΜ Vemurafenib for 3hrs, then assay for in-cell-western and graphed as in A-D. Data shown is the average from two independent experiments.

[0014] Fig. 2A-2E. Ponatinib is an inhibitor of BRAF, while Hesperadin is an inhibitor of MEK. A) BRAF kinase domain activity assay (Millipore) in the absence or presence of Hesperadin, Ponatinib or Vemurafenib, then assay for western blot with the indicated antibodies. B) MEK1 kinase activity assay (Millipore) in the absence or presence of Hesperadin, Ponatinib or Trametinib, then assay for western blot with the indicated antibodies. C) BRAF immunoprecipitated from A375 (BRAF-V600E) mutant melanoma cells and SKMEL239 C4 (p61-BRAF-V600E) resistant melanoma cells was assay for kinase activity in the absence or presence of Hesperadin or Ponatinib, follow by western blot with the indicated antibodies. D-E) Ponatinib potently inhibits kinase activity of both BRAF and CRAF. D) Recombinant BRAF or E) recombinant CRAF kinase domain activity on MEK substrate in the absence or presence of Ponatinib or Vemurafenib, then assay for western blot with the indicated antibodies.

[0015] Fig. 3A-3F. Inhibition of the ERK signaling pathway by Ponatinib and Hesperadin in cells with different RAS/RAF mutational backgrounds. A-B) Melanoma cells A375 (BRAF V600E), SKMEL239 C4 (p61 -BRAF-V600E), SKMEL30 (N-RAS), SKMEL2 (N-RAS) and lung cancer cell CALU6 (K-RAS) were left untreated or treated with (A) Ι μΜ Ponatinib or (B) Ι μΜ Hesperadin for lhr, then assay for western blot and immunoblot with the indicated antibodies. C-E) Dose titration of Vemurafenib and Ponatinib for lhr in RAS mutant cells (C) SKMEL30, (D) SKMEL2 and (E) CALU6 then assay for western blot and immunoblot with the indicated antibodies. F) Ponatinib, not Hesperidin, enhances the formation of BRAF-MEK complex. Melanoma cells SKMEL30 (left panel) and SKMEL2 (right panel) were left untreated or treated 1 μΜ Ponatinib or 1 μΜ Hesperadin for lhr, then cells were collected, assay for MEK1 immunoprecipitation and immunoblot with the indicated antibodies.

[0016] Fig. 4A-4I. Effect of Ponatinib and Hesperadin treatment in cell survival. A)-

F) : Ponatinib and Hesperadin potently induce growth inhibition in various cancer cell lines expressing either BRAF V600E or RAS mutant/BRAF WT. % A) Viability of melanoma cells SKMEL239 C4 (p61-BRAF-V600E), WM266-4 (BRAF V600D), B) A375 (BRAF V600E), SKMEL28 (BRAFV600E), C) A2058 (BRAFV600E), SKMEL30 (N-RAS Q61K/BRAF WT) and D) SKMEL2 (N-RAS Q61R/BRAF WT); colorectal cancer cells D) RKO (BRAF V600E) and E) HT29 (BRAF V600E); pancreatic cancer cells E) Mia-PaCa2 (KRASG12C/BRAF WT) and F) AsPCl (KRAS G12D/BRAF WT); and F) lung cancer cells CALU6 (KRAS G61K/BRAF WT) treated with increasing concentration of Ponatinib or Hesperadin for 72hrs, then measured for cell viability using Cell-Titer Glo (Promega).

G) -I): G) Melanoma cells A375 (BRAF-V600E), SKMEL239 C4 (p61-BRAF-V600E), H) SKMEL30 (N-RAS), SKMEL2 (N-RAS) and I) lung cancer cell CALU6 (K-RAS) were treated with increasing concentration of Ponatinib (left column of each pair) or Hesperadin (right column of each pair) for 24hrs, then measure caspase3/7 activity (Promega).

DETAILED DESCRIPTION OF THE INVENTION

[0017] This invention provides a method of treating a cancer, such as a melanoma or a tumor, in a subject in need thereof comprising administering to the subject ponatinib and/or hesperadin in an amount effective to treat a cancer in a subject.

[0018] The invention provides a method of treating a melanoma in a subject in need thereof comprising administering to the subject ponatinib and/or hesperadin in an amount effective to treat a melanoma in a subject.

[0019] The invention provides a method of treating a tumor in a subject in need thereof comprising administering to the subject ponatinib and/or hesperadin in an amount effective to treat a tumor in a subject. The tumor can be. for example, a thyroid, colon, rectal, lung, pancreatic or hair cell leukemia tumor. Tumors with highest incidence of BRAF mutations include thyroid, colon and hair cell leukemia tumors, and tumors with highest incidence of RAS mutations include the tumors mentioned plus lung and pancreatic tumors.

[0020] Preferably, the subject being treated has been diagnosed as having a melanoma or a tumor.

[0021] The invention further provides a composition comprising ponatinib and/or hesperadin in an amount effective to treat a melanoma or a tumor in a subject and a pharmaceutically acceptable carrier.

[0022] As used herein, the term "treat" a melanoma or a tumor means to reduce the size of the melanoma or tumor, to eradicate the melanoma or tumor, to stabilize the melanoma or tumor so that it does not increase in size or metastasize, or to reduce the further growth of the melanoma or tumor.

[0023] Ponatinib has the structure

and hesperadin has the structure

[0024] Iclusig® (ponatinib) (ARIAD Pharmaceuticals, Inc.) is a kinase inhibitor indicated for the treatment of adult patients with T315I-positive chronic myeloid leukemia (CML) (chronic phase, accelerated phase, or blast phase) or T315I-positive Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL).

[0025] Preferably, ponatinib acts as a RAF inhibitor and hesperadin acts as a MEK inhibitor. These inhibitors were previously developed as BCR-ABL and Aurora inhibitors, respectively. [0026] Ponatinib can be administered in combination with trametinib and/or combimetinib (current FDA approved MEK inhibitors). Hesperadin can be administered in combination with vemurafenib and/or dabrafenib (current FDA-approved RAF inhibitors).

[0027] In one embodiment of the present invention, the subject has not been diagnosed with chronic myeloid leukemia or acute lymphoblastic leukemia. In one embodiment, the subject is not being treated for chronic myeloid leukemia or acute lymphoblastic leukemia with ponatinib.

[0028] The subject can be a mammal. In different embodiments, the mammal is a mouse, a rat, a cat, a dog, a horse, a sheep, a cow, a steer, a bull, livestock, a primate, a monkey, or preferably a human.

[0001] As used herein, the term "carrier" encompasses any of the standard pharmaceutical carriers, such as a sterile isotonic saline, phosphate buffered saline solution, water, and emulsions, such as an oil/water emulsion. The invention also provides pharmaceutical compositions for treating melanoma in a subject comprising any of the compounds disclosed herein and a pharmaceutically acceptable carrier. Examples of acceptable pharmaceutical carriers include, but are not limited to, additive solution-3 (AS- 3), saline, phosphate buffered saline, Ringer's solution, lactated Ringer's solution, Locke- Ringer's solution, Krebs Ringer's solution, Hartmann's balanced saline solution, and heparinized sodium citrate acid dextrose solution. The compound can be administered to the subject in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier used can depend on the route of administration. The pharmaceutical composition can be formulated for administration by any method known in the art, including but not limited to, oral administration, parenteral administration, intravenous administration, transdermal administration and administration through an osmotic mini-pump.

[0002] The compounds and compositions of the present invention can be administered to subjects using routes of administration known in the art. The administration can be systemic or localized to a specific site. Routes of administration include, but are not limited to, intravenous, intramuscular, intrathecal or subcutaneous injection, oral or rectal or transdermal administration, and injection into a specific site.

[0003] Also provided is the use of ponatinib and/or hesperadin as a medicament for treatment of a cancer such as a melanoma or a tumor. [0004] The invention also provides an in-cell-western based screening assay for identifying candidate compounds for treatment of melanoma or a tumor comprising

measuring fluorescence levels of phosphorylated ERK (P-ERK) in melanoma or tumor cells harboring a constitutively active splice variant p61-BRAF^V600E dimer that is resistant to the BRAF inhibitor vemurafenib;

contacting the cells with the compound in the absence of vemurafenib; and identifying the compound as a candidate compound for treatment of melanoma or a tumor if the compound reduces P-ERK fluorescence levels to 50% or less in the absence of vemurafenib.

[0029] The melanoma cells used in the assay can be, for example, A375, SKMEL30, SKMEL2, SKMEL-28, A2058, WM-266-4 or SKMEL239 C4 melanoma cells. The tumor cells used in the assay can be, for example, colorectal cancer cells RKO (BRAF V600E), colorectal cancer cells HT29 (BRAF V600E), pancreatic cancer cells Mia-PaCa2 (KRASGl 2C/BRAF), pancreatic cancer cells AsPC l (KRAS G12D/BRAF), or lung cancer cells CALU6 (KRAS G61K/BRAF).

[0030] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims, which follow thereafter.

EXPERIMENTAL DETAILS

Introduction

[0031] The MAPK cascade consists of activation of RAS proteins that stimulate the RAF kinases ARAF, BRAF, and CRAF. This process causes the activation of the MEK kinases, which activate the ERK kinases. Activated ERK kinases then regulate multiple biological processes involved in cell survival. BRAF mutations are one of the most common mutations in melanoma. The most frequent mutation is the substitution of valine at position 600 for glutamic acid, known as BRAF^V600E. Current RAF FDA-approved drugs target these BRAF mutation form. However, melanomas with other types of mutations are refractory to these RAF inhibitors. To overcome this, there is one FDA-approved allosteric MEK inhibitor, but its toxicity makes it a limited agent. In contrast to current RAF FDA- approved drugs, a new RAF inhibitor is identified herein that can block the MAPK pathway in melanoma cells with different RAS/RAF mutational backgrounds. Also identified is a new MEK inhibitor, which in contrast to most current MEK inhibitors developed today, is an ATP competitive MEK inhibitor.

Experimental Procedures

[0032] Cell culture, Cell viability assay and Cell caspase activity assay. A375, SKMEL30 and SKMEL2 melanoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 1% Pen-Strep, and 1% Glutamine. SKMEL239 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 1% Pen-Strep, and 1% Glutamine in the presence of ΙμΜ Vemurafenib (PLX 4032 from LC Laboratories). CALU6 cells were grown in Roswell Park Memorial Institute medium (RPMI) with 10% fetal bovine serum (FBS), 1% Pen-Strep, and 1% Glutamine. For cell viability assays and caspase 3/7 activity, manufacture protocols Cell-Titer Glo (Promega) and Caspase-Glo 3/7 Assay (Promega) were followed, respectively.

[0033] In-Cell-Western screening. SKMEL239 clone 4 melanoma cells were plated in 96-well plate in DMEM 10% FBS, 1% Pen-Strep and 1% Glutamine, and allowed to seed overnight. Media was removed and replaced with fresh media containing 0.5μΜ Vemurafenib (PLX4032) and treated with 5μΜ of corresponding kinase inhibitors and incubated for 3hrs. Cells were then fixed in 4% formaldehyde for 20min at room temperature (RT), and washed 4 times with 0.1% Triton in IX PBS for 5 minutes at RT with gentle rocking. Cells were then rinsed with IX PBS and stored in IX PBS at 4°C for future in-cell-western (ICW). For ICW, LiCor PI-140 0103 Doc #988-07083 protocol was followed with some modifications. In brief, cells were blocked with Odyssey blocking solution (LiCor) for lhr at RT. Then cells were incubated with primary antibodies diluted in odyssey blocking buffer (1 :200 P-ERK1 and 1 :200 ERK1) for 2hrs at RT, followed by 4 washes with 0.1% Tween 20 in IX PBS (PBST) for 5 min at RT. Then cells were incubated for 2hrs at RT with secondary antibodies diluted in odyssey blocking buffer containing 0.2% Tween-20 (1 :800 IRDye800CW anti-mouse and 1 :800 IRDye680RD anti-rabbit), and washed 4 times with PBST for 5min at RT. Then rinsed once with IX PBS, aspirated off and imaged with Odyssey Classic imager (ODY-0671).

[0034] Western blotting, co-immpunoprecipitation and kinase assays. Western blots were performed from whole cell lysates (WCL) prepared in lysis buffer containing 50mM Tris-HCl pH7.5, 1% NP40, 150mM NaCl, lmM EDTA, and 10% glycerol in the presence of protease inhibitor cocktail (Roche). WCL were separated on a 4-12% NuPAGE MES gel (Invitrogen), transferred into a PVDF membrane, blocked for lhr and immunoblotted with the corresponding antibodies.

[0035] Co-immunoprecipitation assays were performed from whole cell lysate prepared in lysis buffer in the presence of protease inhibitor cocktail (Roche) and incubated at 4°C overnight with gentle rotation, then protein G beads were added and incubated for 2hrs more at 4°C. Kinase activity assay were performed following the manufacturer's protocol with some modifications (Millipore).

[0036] Antibodies. BRAF (Santa Cruz sc-5284), CRAF (Santa Cruz C-12) MEK1 (Millipore), MEK1/2 (Cell Signaling 4694), P-Mekl/2 (Cell Signaling 9154), ERK1/2 (Cell Signaling 4696), ERK1 (Santa Cruz sc-7383), P-ERK1/2 (Cell Signaling 4370), P-ERK1/2 (Cell Signaling 9101), and Actin (Invitrogen) were used.

Results

[0037] In-cell-western-based drug screening of kinase inhibitors. In order to find novel inhibitors of the RAS/RAF/MEK/ERK signaling pathway that can overcome resistance to current drugs in melanoma, an in-cell-western based screen assay was conducted in SKMEL239 C4 melanoma cells harboring a constitutively active splice variant p61-BRAF^V600E dimer that is resistant to the BRAF inhibitor vemurafenib (PLX4032).²¹ In this cell-based screen assay, fluorescence levels of phosphorylated ERK (P-ERK), the downstream effector of the RAS/RAF/MEK/ERK signaling cascade, were measured as a read out for inhibition. First, the efficiency of the in-cell-western assay was tested in measuring P-ERK fluorescence levels in cells treated with vemurafenib and trametinib (MEK inhibitor). For this, SKMEL239 C4 melanoma cells were treated with increasing concentration of each inhibitor (Figure 1A-1C). As expected, only high doses of vemurafenib reduced the levels of P-ERK fluorescence (Figure 1A-1B).²¹ On the contrary, trametinib decreased P-ERK fluorescence levels at much lower doses (Figure 1C). Trametinib targets MEK, which acts downstream the p61-BRAF^V600E constitutively active dimer that drives the cascade signaling in these resistant melanoma cells. Hence, lower doses of trametinib have a stronger effect than current RAF inhibitors on these cells. Although MEK inhibitors are more potent than RAF inhibitors, they still need to be combined with RAF inhibitors to reach maximum inhibition of the ERK signaling pathway.²²

[0038] Next the suitability of the in-cell-western assay for use in a full-scale and high- throughput screen was quantified. For this, a measure of statistical effect size termed Z- factor was calculated. Vemurafenib (0.5μΜ) was used as a negative control (the non- inhibitory condition) and 0.1 μΜ trametinib was used as a positive control (inhibitory condition), which resulted in a Z-factor of 0.5, which correlates with an excellent assay, in which 0.5 is equivalent to a separation of 12 standard deviations between the negative and positive control.

[0039] Upon optimization and validation of the in-cell-western assay, kinase inhibitors were screened that in combination with vemurafenib could restore cell sensitivity and reduce the levels of P-ERK in cells. From this screen approximately 77 kinase inhibitors were identified, which reduced P-ERK fluorescence levels to 50% or less (Figure ID). Next a second screen was conducted (with the hits obtained in the initial screen) in the absence or presence of vemurafenib. The goal of this second screen was to identify: 1) those kinase inhibitors that reproduce the results from the first screen in the presence of vemurafenib, 2) kinase inhibitors that alone did not reduce phosphorylation of ERK (absence of vemurafenib) and 3) novel kinase inhibitors that alone are very potent inhibitors of P-ERK fluorescence levels (Figure IE). From this second screen, 11 potent kinase inhibitors were obtained, which alone (absence of vemurafenib) reduced P-ERK fluorescence levels to 50% or less (Figure IE). Among the hits identified, Ponatinib and Hesperadin had the strongest inhibition of the ERK signaling driven by the p-61 -BRAF^V600E constitutively active dimer expressed in SKMEL239 C4 melanoma cells (Figure 2C). Therefore, it was decided to further investigate the mechanism of ERK signaling inhibition by Ponatinib and Hesperadin.

[0040] Identification of Ponatinib as a BRAF inhibitor and Hesperadin as a MEK inhibitor. Having identified Ponatinib and Hesperadin as novel inhibitors of the p61- BRAF^V600E driven ERK signaling, their ability to inhibit BRAF kinase activity was examined. To address this, recombinant BRAF protein kinase domain was incubated in the presence of increasing concentration of Ponatinib or Hesperadin for 15min and then inactive MEK1 was added as substrate (Figure 2A). Interestingly, Ponatinib inhibited BRAF kinase domain activity in vitro while Hesperadin did not (Figure 2A). These results indicate two different inhibitory mechanisms of the p61-BRAF^V600E driven ERK signaling pathway by each kinase inhibitor. Hesperadin was hypothesized to inhibit downstream p61-BRAF^V600E signaling perhaps through inhibition of MEK instead. To test this, recombinant MEK1 protein was incubated in the presence of increasing concentrations of Ponatinib or Hesperadin, and then inactive ERK2 was added as substrate. In this setting, Ponatinib did not inhibit MEKl kinase activity, while Hesperadin did, validating the hypothesis. Next, it was examined whether Ponatinib could inhibit BRAF^V600E active monomer and splice variant p61 -BRAF^V600E constitutively active dimer immunoprecipitated from cells. As before, immunoprecipitated proteins were incubated with Ponatinib or Hesperadin for 15min and then inactive MEKl was added (Figure 2C). Ponatinib inhibited kinase activity of BRAF^V600E and p61-BRAF^V600E, while Hesperadin did not. Taken together these results indicate that Ponatinib is a new BRAF inhibitor and Hesperadin is a new MEK inhibitor.

[0041] Paradoxical activation by Ponatinib. Until now the kinase inhibitors screen and analysis had been conducted in p61 -BRAF^V600E splice variant resistant melanoma cells. Therefore, inhibition of the ERK signaling pathway by Ponatinib and Hesperadin was also tested in cells with other RAF/RAS mutational background (Figure 3A & 3B). For this, A375 (BRAF^V600E mutant), SKMEL30 and SKMEL2 (NRAS mutant/BRAF WT) and CALU6 (KRAS mutant/BRAF WT) cells lines were used. Treatment with Ι μΜ Hesperadin reduced phosphorylation of ERK in all the cell lines (Figure 3B). However, Ponatinib at 1 μΜ dose only reduced phosphorylation of ERK on A375 and SKMEL239 C4, while it increased phosphorylation of ERK in SKMEL30 and SKMEL2, and had no effect on CALU6 (Figure 3 A). Perhaps on RAS mutant cells Ι μΜ Ponatinib is not an optimal dose for inhibiting the RAS/RAF/MEK/ERK signaling. Hence a dose titration of Ponatinib was conducted to find the optimal dose for inhibiting phosphorylation of ERK (Figure 3C-3E). Treatment of RAS mutant cells lines with Ponatinib at doses of 1 μΜ or less led to increase phosphorylation of MEK and ERK. In order to reduce phosphorylation of MEK and phosphorylation of ERK a dose of at least 3μΜ was necessary; still Ponatinib had a better inhibition than vemurafenib in these cells (Figure 3C-3E). Although Ponatinib at Ι μΜ inhibits BRAF and reduces phosphorylation of MEK and ERK in A375 and SKMEL239 C4, a dose titration experiment was conducted in these cells as well. Interestingly, in A375 cells vemurafenib is a better inhibitor than Ponatinib. Vemurafenib reduced P-ERK and P- MEK levels at 0.1 μΜ dose while Ponatinib does it at 0.5μΜ dose. On the contrary, in SKMEL239 C4, Ponatinib has better inhibition than Vemurafenib.

[0042] RAF regulation is highly complex and it involves several activation events.²' ⁵' ⁶ One key step in the activation of RAF is the formation of homo and heterodimers within the RAF members.⁸ RAF dimer formation is necessary for wild type RAF kinase activity, because RAF monomer is inactive, as several studies have suggested.⁵ Mutations in the dimer interface or peptide inhibitor of the dimer interface can block dimerization and function of RAF proteins with low or no monomeric kinase activity. ' While BRAF can signal as a monomer, wild type BRAF requires RAS dependent dimerization for its kinase activity.²¹' ²⁴ Therefore, it was examined whether Ponatinib could disrupt RAS dependent dimerization of RAF proteins as part of its mechanism of BRAF inhibition. For this, RAS mutant cells were treated with the non-inhibitory dose of Ι μΜ and an inhibitory dose of 5μΜ of Ponatinib. Interestingly, Ponatinib enhance BRAF-CRAF heterodimer formation at both doses. Although, it enhance dimerization of BRAF-CRAF heterodimers at 5uM it still reduced phosphorylation of ERK, suggesting Ponatinib can inhibit RAF dimers. This was also confirmed in BRAF^V600E and p61-BRAF^V600E cell lines. Ponatinib inhibits BRAF to reduced phosphorylation of ERK and phosphorylation of MEK, while inducing BRAF-CRAF heterodimer formation.

[0043] In summary, an in-cell-western based screen assay was used to identify kinase inhibitors of the ERK signaling pathway. Both candidates inhibit the ERK signaling pathway by targeting two different components of the ERK signaling cascade, BRAF and MEK. Ponatinib inhibits BRAF kinase activity, while Hesperadin inhibits MEK kinase activity. In addition, both candidates decrease cell survival in cells with RAS/RAF different mutational in contrast to the FDA-approved drug Vemurafenib (Fig. 4). Ponatinib is currently an FDA approved drug developed by ARIAD Pharmaceuticals for the treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). It is a multitargeted tyrosine-kinase inhibitor. The present results indicate that Ponatinib can also be used for the treatment of melanoma in patients that are intrinsically resistant or have developed resistance to current clinical treatments. Ponatinib treatment could potentially serve as a novel alternative treatment for melanoma and other cancers in which the RAS/RAF/MEK/ERK pathway is abnormally regulated. The present results have shown that Ponatinib can inhibit the ERK signaling pathway driven by RAS and RAF mutations. Hesperadin is an aurora kinase inhibitor not developed or used for any therapeutic purposes today. The present results indicate that it can serve as a therapy for the treatment of melanoma as well.

REFERENCES

1. De Luca, A.; Maiello, M. R. ; D'Alessio, A. ; Pergameno, M. ; Normanno, N., The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets 2012, 16 Suppl 2, S I 7-27. 2. Wellbrock, C; Karasarides, M.; Marais, R., The RAF proteins take centre stage. Nat Rev Mol Cell Biol 2004, 5 (11), 875-85.

3. Schubbert, S.; Bollag, G.; Shannon, K., Deregulated Ras signaling in developmental disorders: new tricks for an old dog. Curr Opin Genet Dev 2007, 17 (1), 15-22; Schubbert, S.; Shannon, K.; Bollag, G., Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer 2007, 7 (4), 295-308; Shaw, R. I; Cantley, L. C, Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 2006, 441 (7092), 424-30.

4. Zebisch, A.; Troppmair, J., Back to the roots: the remarkable RAF oncogene story. Cell Mol Life Sci 2006, 63 (11), 1314-30.

5. Roskoski, R., RAF protein-serine/threonine kinases: structure and regulation.

Biochem Biophys Res Commun 2010, 399 (3), 313-7.

6. Wittinghofer, A.; Nassar, N., How Ras-related proteins talk to their effectors. Trends Biochem Sci 1996, 21 (12), 488-91.

7. Maurer, G; Tarkowski, B.; Baccarini, M., Raf kinases in cancer-roles and therapeutic opportunities. Oncogene 2011, 30 (32), 3477-88.

8. Rushworth, L. K.; Hindley, A. D.; O'Neill, E.; Kolch, W., Regulation and role of Raf-l/B-Raf heterodimerization. Mol Cell Biol 2006, 26 (6), 2262-72; Wimmer, R; Baccarini, M., Partner exchange: protein-protein interactions in the Raf pathway. Trends Biochem Sci 2010, 35 (12), 660-8.

9. Montagut, C; Settleman, J., Targeting the RAF-MEK-ERK pathway in cancer therapy. Cancer Lett 2009, 283 (2), 125-34; Pritchard, C. A.; Samuels, M. L.; Bosch, E.; McMahon, M., Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells. Mol Cell Biol 1995, 15 (11), 6430-42.

10. Freeman, A. K.; Ritt, D. A.; Morrison, D. K., Effects of raf dimerization and its inhibition on normal and disease-associated raf signaling. Mol Cell 2013, 49 (4), 751-8.

11. Davies, H.; Bignell, G. R.; Cox, C; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. I; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C; Parker, A.; Stevens, C; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench, G; Riggins, G. J.; Bigner, D. D.; Palmieri, G; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R; Futreal, P. A., Mutations of the BRAF gene in human cancer. Nature 2002, 417 (6892), 949-54.

12. Pratilas, C. A.; Taylor, B. S.; Ye, Q.; Viale, A.; Sander, C; Solit, D. B.; Rosen, N., (V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway. Proc Natl Acad Sci U S A 2009, 106 (11), 4519-24.

13. Bollag, G.; Tsai, I; Zhang, I; Zhang, C; Ibrahim, P.; Nolop, K.; Hirth, P., Vemurafenib: the first drug approved for BRAF-mutant cancer. Nat Rev Drug Discov 2012, 11 (11), 873-86.

14. Chappell, W. H.; Steelman, L. S.; Long, J. M.; Kempf, R. C; Abrams, S. L.; Franklin, R. A.; Basecke, J.; Stivala, F.; Donia, M.; Fagone, P.; Malaponte, G.; Mazzarino, M. C; Nicoletti, F.; Libra, M.; Maksimovic-Ivanic, D.; Mijatovic, S.; Montalto, G; Cervello, M.; Laidler, P.; Milella, M.; Tafuri, A.; Bonati, A.; Evangelisti, C; Cocco, L.; Martelli, A. M.; McCubrey, J. A., Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: rationale and importance to inhibiting these pathways in human health. Oncotarget 2011, 2 (3), 135-64.

15. Poulikakos, P. I.; Zhang, C; Bollag, G; Shokat, K. M.; Rosen, N., RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 2010, 464 (7287), 427-30.

16. Hatzivassiliou, G; Song, K.; Yen, I.; Brandhuber, B. J.; Anderson, D. J.; Alvarado, R.; Ludlam, M. J.; Stokoe, D.; Gloor, S. L.; Vigers, G; Morales, T.; Aliagas, I.; Liu, B.; Sideris, S.; Hoeflich, K. P.; Jaiswal, B. S.; Seshagiri, S.; Koeppen, H.; Belvin, M.; Friedman, L. S.; Malek, S., RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 2010, 464 (7287), 431-5.

17. Heidorn, S. J.; Milagre, C; Whittaker, S.; Nourry, A.; Niculescu-Duvas, I.; Dhomen, N.; Hussain, J.; Reis-Filho, J. S.; Springer, C. J.; Pritchard, C; Marais, R., Kinase- dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 2010, 140 (2), 209-21.

18. Lemech, C; Infante, J.; Arkenau, H. T., Combination molecularly targeted drug therapy in metastatic melanoma: progress to date. Drugs 2013, 73 (8), 767-77.

19. Shi, H.; Hugo, W.; Kong, X.; Hong, A.; Koya, R. C; Moriceau, G; Chodon, T.; Guo, R; Johnson, D. B.; Dahlman, K. B.; Kelley, M. C; Kefford, R. F.; Chmielowski, B.; Glaspy, J. A.; Sosman, J. A.; van Baren, N.; Long, G. V.; Ribas, A.; Lo, R. S., Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov 2014, 4 (1), 80-93.

20. Nazarian, R.; Shi, H.; Wang, Q.; Kong, X.; Koya, R. C; Lee, H.; Chen, Z.; Lee, M. K.; Attar, N.; Sazegar, H.; Chodon, T.; Nelson, S. F.; McArthur, G.; Sosman, J. A.; Ribas, A.; Lo, R. S., Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N- RAS upregulation. Nature 2010, 468 (7326), 973-7.

21. Poulikakos, P. I.; Persaud, Y.; Janakiraman, M.; Kong, X.; Ng, C; Moriceau, G.; Shi, H.; Atefi, M; Titz, B.; Gabay, M. T.; Saltan, M.; Dahlman, K. B.; Tadi, M.; Wargo, J. A.; Flaherty, K. T.; Kelley, M. C; Misteli, T.; Chapman, P. B.; Sosman, J. A.; Graeber, T. G; Ribas, A.; Lo, R. S.; Rosen, N.; Solit, D. B., RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 2011, 480 (7377), 387-90.

22. Flaherty, K. T.; Infante, J. R.; Daud, A.; Gonzalez, R.; Kefford, R. F.; Sosman, J.; Hamid, O.; Schuchter, L.; Cebon, J.; Ibrahim, N.; Kudchadkar, R.; Burris, H. A.; Falchook, G; Algazi, A.; Lewis, K.; Long, G. V.; Puzanov, I.; Lebowitz, P.; Singh, A.; Little, S.; Sun, P.; Allred, A.; Ouellet, D.; Kim, K. B.; Patel, K.; Weber, J., Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med 2012, 367 (18), 1694- 703.

23. Zhang, J. H.; Chung, T. D.; Oldenburg, K. R., A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 1999, 4 (2), 67-73.

24. Roring, M.; Herr, R.; Fiala, G. J.; Heilmann, K; Braun, S.; Eisenhardt, A. E.; Halbach, S.; Capper, D.; von Deimling, A.; Schamel, W. W.; Saunders, D. N.; Brummer, T., Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling. EMBO J 2012, 31 (11), 2629-47.

25. Rigel DS Malignant melanoma: incidence issues and their effect on diagnosis and therapy in the 1990s. Mayo Clin. Proa, 72: 367-371, 1997.

26. Grin-Jorgensen CM, Rigel DS and Friedman RJ The world-wide incidence of malignant melanoma. In: CM. Balch, A.N. Houghton, G.W. Milton, A.J. Sober, and S.J. Soong (eds.), Cutaneous Melanoma, Ed. 2, pp. 27-39. Philadelphia: J.B. Lippincott Co., 1992.

27. Liu T and Soong SJ Epidemiology of malignant melanoma. Surg. Clin. N. Am., 76: 1205-1222, 1996. 28. Sun W, Schuchter LM. Metastatic melanoma. Curr Treat Options Oncol 2: 193-202, 2001.

Claims

What is claimed is:

1. A method of treating a cancer in a subject in need thereof comprising administering to the subject ponatinib and/or hesperadin in an amount effective to treat a cancer in a subject.

2. The method of claim 1 , where in the cancer is melanoma.

3. The method of claim 1 , where in the cancer is a tumor.

4. The method of claim 3, wherein the tumor is selected from the group consisting of thyroid, colon, rectal, lung, pancreatic and hair cell leukemia tumors.

5. The method of any of claims 1 -4, wherein the subject has been diagnosed as having a melanoma or a tumor.

6. The method of any of claims 1 -4, wherein the subject does not have and is not being treated for chronic myeloid leukemia or acute lymphoblastic leukemia.

7. The method of any of claims 1 -4, wherein ponatinib acts as a RAF inhibitor.

8. The method of any of claims 1 -4, wherein hesperadin act as a MEK inhibitor.

9. The method of any of claims 1 -4, wherein ponatinib is administered in combination with trametinib and/or combimetinib.

10. The method of any of claims 1-4, wherein hesperadin is administered in combination with vemurafenib and/or dabrafenib.

1 1. The method of any of claims 1 -10, wherein the subject is a mammal.

12. The method of claim 1 1, wherein the mammal is a human.

13. A composition comprising ponatinib and/or hesperadin in an amount effective to treat a melanoma or a tumor in a subject and a pharmaceutically acceptable carrier.

14. An in-cell-western based screening assay for identifying candidate compounds for treatment of a melanoma or a tumor comprising

contacting the cells with the compound in the absence of vemurafenib; and

identifying the compound as a candidate compound for treatment of melanoma or a tumor if the compound reduces P-ERK fluorescence levels to 50% or less in the absence of vemurafenib.

15. The assay of claim 14, wherein the melanoma cells are A375, SKMEL30, SKMEL2, SKMEL-28, A2058, WM-266-4 or SKMEL239 C4 melanoma cells.

16. The assay of claim 14, wherein the melanoma cells are SKMEL239 C4 melanoma cells.

17. The assay of claim 14, wherein the tumor cells are colorectal cancer cells RKO (BRAF V600E), colorectal cancer cells HT29 (BRAF V600E), pancreatic cancer cells Mia- PaCa2 (KRASG12C/BRAF), pancreatic cancer cells AsPCl (KRAS G12D/BRAF), or lung cancer cells CALU6 (KRAS G61K/BRAF).